Characterization of human umbilical cord mesenchymal stem cells following tissue mass culture.

The human umbilical cord represents a promising resource of mesenchymal stem cells (MSCs). In order to improve our understanding of MSCs derived from human umbilical cord (UC-MSCs), we isolated UC-MSCs from human umbilical cord tissues through a direct culture approach. We performed a comprehensive characterization of these cells based on analyses of morphology, growth features, cell surface antigen markers and differentiation capacity. All these analyses validated their stem cell nature. The UC-MSCs presented a spindle-shaped morphology and could be subcultured for up to 15 passages without losing their cellular features. Moreover, these UC-SMCs presented an expression profile of cell surface antigens similar to other MSCs: positive for CD44, CD90, and CD105 expression and negative for CD34, CD31, and CD45 expression. Differentiation assays further validated the multipotency of UC-MSCs by inducing these cells into osteoblasts, adipocytes and functional hepatocytes. Our studies clearly demonstrated that UC-MSCs resemble other types of MSCs in many aspects and have a great potential to be applied in tissue engineering and regenerative medicine.

Cortical interneurons become activated by deafferentation and instruct the apoptosis of pyramidal neurons.

Unlike peripheral nervous system neurons and certain groups of nerve cells in the CNS, cortical projection neurons are tolerant of axonal lesions. This resistance is incongruent with the massive death of pyramidal neurons in age-associated neurodegenerative diseases that proceed along corticocortical connections. Some insights have emerged from our previous work showing that pyramidal cells in piriform cortex undergo classical apoptosis within 24 h after bulbectomy via transsynaptic, but not retrograde, signaling. These findings allow the investigation of cellular and molecular changes that take place in the context of experimental cortical degeneration. In the present study, we show that the transsynaptic death of pyramidal neurons in piriform cortex is a nitric oxide-mediated event signaled by activated interneurons in layer I. Thus, we demonstrate that cortical interneurons play an essential role in transducing injury to apoptotic signaling that selectively targets pyramidal neurons. We propose that this mechanism may be generic to cortical degenerations and amenable to therapeutic interventions.

The preparation and characterization of an antimicrobial polypeptide from the loach, Misgurnus anguillicaudatus.

Using Sephadex G-50 gel filtration, DEAE-52 cellulose ion-exchange chromatography, and an improved polyacrylamide gel electrophoresis together with electroelution, a novel polypeptide with antimicrobial activity in vitro was isolated and characterized from loach, Misgurnus anguillicaudatus. The polypeptide, named MAPP, contains about 94 residues containing l0 different amino acids, of which cysteine was the most abundant. No alkaline residue was found in MAPP. MAPP is a single-chain polypeptide with Mw of about 9800Da and pI of about 4.78; the N-terminus of MAPP was CFGWN. MAPP showed good inhibition of various bacteria including Bacillus subtilis, Escherichia coli, and Staphylococcus aureus. MAPP is thermally stable with more than 70% inhibitory bioactivity remaining after treatment at 60 degrees C for 30min. In addition, MAPP could inhibit the autoxidation of pyrogallol with a high efficiency. Similarity searches by comparing amino acid composition, MS-fingerprint, and the N-terminus of MAPP demonstrated that no protein exactly matched MAPP in databases around the world.

Isolation and partial characterization of selenium-containing tRNA from germinating barley.

Selenium-containing tRNA was discovered in germinating barley for the first time with the 75Se isotopic tracer technique; therefore, this technique was used to study the effect of different concentrations of selenium and sulfur in the medium on the content of selenium-containing tRNA in germinating barley. Se-containing tRNAs and its hydrolysates were isolated, purified, and characterized by means of column chromatography, ion-exchange chromatography, high-performance liquid chromatography, and the ultraviolet-visible spectrum. The results show that the amount of selenium in tRNA is almost unaffected by the sulfuric content in the medium, and the pathway for selenium and sulfur to enter tRNA might not be exactly the same. Selenium exists within tRNA in the form of 5-methylamine methyl-2-selenouridine, just as it does within a microorganism tRNA.

Identification and characterization of a second butyrate kinase from Clostridium acetobutylicum ATCC 824.

A gene encoding a new butyrate kinase isozyme (BKII) was identified from the C. acetobutylicum ATCC 824 DNA database. The enzyme was expressed in Escherichia coli, purified, and characterized. The purified enzyme exhibited a subunit molecular mass of 43 kDa by SDS-PAGE, and a native molecular mass of 80 kDa by gel filtration suggesting it functions as a dimer. In the butyryl phosphate-forming direction the optimal pH of BKII was 8.5. The enzyme had a Km of 0.62 M and a turn over rate of 2.2 x 10(5)/sec (Vmax of 165 units/mg). The presence of a mRNA encoding the BKII was demonstrated using a reverse transcription PCR reaction. The expression of the BKII in Clostridium acetobutylicum ATCC 824 was further examined by Western blot analysis using a polyclonal antibody prepared against recombinant BKII.

Ser67-phosphorylated inhibitor 1 is a potent protein phosphatase 1 inhibitor.

Inhibitor 1 (I-1) is a protein inhibitor of protein phosphatase 1 (PP1), a major eukaryotic Ser/Thr phosphatase. Nonphosphorylated I-1 is inactive, whereas phosphorylated I-1 is a potent PP1 inhibitor. I-1 is phosphorylated in vivo on Thr(35) and Ser(67). Thr(35) is phosphorylated by cAMP-dependent protein kinase (A kinase), and Thr(35)-phosphorylated I-1 inhibits PP1. Until now the kinase that phosphorylates Ser(67) had not been identified and the physiological role of Ser(67) phosphorylation was unknown. In this study we detected a high level of kinase activity in brain extract when a glutathione S-transferase (GST) fusion I-1 mutant containing an Ala substituted for Thr(35) [GST-I-1(T35A)] was used as the substrate. GST-I-1(T35A) kinase and neuronal cdc2-like protein kinase (NCLK) in the brain extract could not be separated from each other by a series of sequential chromatographies. GST-I-1(T35A) kinase immunoprecipitated with anti-NCLK antibody from kinase-active column fractions. Purified NCLK-phosphorylated GST-I-1(T35A) and I-1 (0.7 mole of phosphate per mole of I-1). HPLC phosphopeptide mapping, amino acid sequencing, and site-directed mutagenesis determined that NCLK phosphorylates Ser(67) of I-1. NCLK-phosphorylated I-1 and I-1(T35A) inhibited PP1 with IC(50) values approximately 9.5 and 13. 8 nM, respectively. When compared, A kinase-phosphorylated I-1 was only approximately 1.2 times more inhibitory than NCLK-phosphorylated I-1. Our data indicate that NCLK is a potential in vivo I-1 kinase and that Thr(35) and Ser(67) phosphorylation independently activate I-1.

Electrotransformation of Clostridium acetobutylicum ATCC 824 using high-voltage radio frequency modulated square pulses.

Molecular biological improvement of industrial solventogenic clostridia could be enhanced by a higher efficiency of electrotransformation. In this research, we used a new approach to determine the frequency spontaneously generated by Clostridium acetobutylicum ATCC 824 cells during the application of a square high-voltage pulse. Once the frequency of 100 kHz was determined we transformed clostridial cells with pSOS84 plasmid DNA using radio-frequency modulated high-voltage square pulses (electric field strength 12 kVcm-1; pulse duration 22.5 ms; frequency of pulse modulation 100 kHz) to reach an efficiency exceeding 106 transformants microg-1 of plasmid DNA. We propose a possible role for cellular membrane structures in affecting the transformation yield.

An external-sample liquid scintillation counting for 75Se and its application to selenoprotein detection.

An external-sample liquid scintillation (LS) counting for the gamma emitter 75Se has been developed. An expressly designed well-type LS vial and a 2,5-diphenyoxazole-1,4-bis(5-phenyl-2-oxazoyl)-benzene-xylene solution containing 35% tertrabutylzinn allow 75Se to be counted in a standard LS counter with counting efficiency up to 43.2%, much higher than that of conventional LS counting method. This external sample LS has a good count rate linearity and exhibits low background count rates. After in vivo labeling with [75Se]selenite, 75Se distributions and the Se-containing proteins present in tissues of male rat were investigated by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, external-sample LS and gamma-detector. Eight Se-containing proteins or protein subunits were detected to be Se-containing proteins or protein subunits in arterial wall, and their apparent molecular masses (Mr) were 76.4, 67.0, 57.4, 30.3, 25.4, 22.7, 21.7, and 15.1 kDa, respectively. In addition, eight 75Se-labeled proteins (Mr: 66.8, 57.0, 43.1, 30.0, 24.8, 19.8, 18.0, and 14.8 kDa) were found in brain homogenates, and nine 75Se-labeled proteins (Mr: 117.0, 78.0, 66.6, 57.2, 43.0, 38.1, 25.0, 20.1, and 18.0 kDa) were detected in testis homogenates. Some of them should be new biologically important selenoproteins that have not been identified so far.

[Studies on chemical constituents of Curcuma aromatica salisb].

OBJECTIVE: To study the effective ingredients of Curcuma aromatica. METHOD: Solvent extraction was used. The constituents were isolated with resin D-101 silica gel column and thin-layer chromatography, and the structures were elucidated by physico-chemical properties and spectral analysis. RESULT: Curdione, neocurdione, curcumol, tetramethylpyrazine and (R)-(+)-1,2-hexadecanediol were isolated from C. aromatica. CONCLUSION: Neocurdione and (R)-(+)-1,2-hexadecanediol were isolated from C. aromatica for the first time, and was isolated from Curcuma and reported for the first time.

Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.

We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside. The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein had a dimeric structure composed of identical subunits, and the M(r) of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit M(r) was 36, 000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K(m)) for (RS)-mevalonate was 68.5 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.

N-methyl-D-aspartate receptor blockade attenuates D1 dopamine receptor modulation of neuronal activity in rat substantia nigra.

It has been proposed that dopamine and glutamate affect basal ganglia output, in part, through interactions between D1 receptors and NMDA receptors. The present study examined whether N-methyl-D-aspartate (NMDA) receptor antagonists affect the neurophysiological responses of substantia nigra pars compacta (SNpc; dopaminergic) and pars reticulata (SNpr; non-dopaminergic) neurons to a systemically administered D1 dopamine agonist in two animals models of Parkinson's disease, reserpine treatment and nigrostriatal lesion. Previous studies using extracellular single unit recording techniques have shown that the D1 dopamine agonist SKF 38393 (10 mg/kg) exerts different effects on the firing rates of SNpr neurons after these two dopamine-depleting treatments, suggesting the involvement of multiple mechanisms. SKF 38393 consistently increased the firing rates of SNpr neurons in rats treated subchronically with reserpine, and markedly decreased SNpr firing rates in rats with nigrostriatal damage. Pretreatment with the non-competitive NMDA antagonist MK-801 (0.15 mg/kg i.v.) blocked, and the competitive NMDA antagonist (+/-)-CPP (30 mg/kg i.p.) attenuated, the rate effects of SKF 38393 in both dopamine-depleted preparations. SKF 38393 consistently inhibited the firing rate of SNpc dopamine neurons after acute reserpine treatment (10 mg/kg, 4-7 hours), an effect specifically mediated by D1 receptors. Pretreatment with MK-801 (0.1 mg/kg i.v.) or the competitive NMDA antagonist (+)-HA-966 (30 mg/kg i.v.) also effectively attenuated SKF 38393's inhibitory effect on SNpc dopamine neurons. Therefore, NMDA receptor blockade markedly reduces the ability of D1 receptor stimulation to modulate firing rates of both dopaminergic and non-dopaminergic cells in the substantia nigra. Although multiple mechanisms appear to underlie D1-mediated effects on substantia nigra firing rates in reserpine and 6-OHDA-treated rats, these results demonstrate a common dependence on glutamatergic transmission and a permissive role for NMDA receptor activation in the ability of D1 receptor stimulation to both enhance and reduce neuronal activity in the substantia nigra.

Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway.

Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadiene synthase was cloned recently. Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein. Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxadiene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a single step with a His-binding metal affinity column. The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.

Dopaminergic regulation of AP-1 transcription factor DNA binding activity in rat striatum.

Dopaminergic modulation of the DNA binding activity of AP-1, Sp1, CREB and AP-2 transcription factors was examined in rat striatal nuclear extracts by gel shift assay. AP-1 binding was selectively increased in the striatum following depletion of dopamine by 6-hydroxydopamine-induced lesion of the nigrostriatal pathway or after reserpine treatment. The D1 agonist SKF 38393 dose-dependently increased AP-1 binding; this effect was significantly increased in reserpine-treated rats and even more markedly enhanced in denervated striatum. The D2/D3 agonist quinpirole, administered alone, did not affect striatal activator protein-1 binding; in combination, quinpirole and SKF 38393 acted synergistically in normal and reserpine-treated rats but not in 6-hydroxydopamine-lesioned rats, suggesting that mechanisms underlying D1-D2/D3 interactions are altered after dopamine denervation. Most, but not all, of the changes in AP-1 binding activity observed in this study are consistent with changes in levels of Fos/Jun family proteins observed after similar treatments. These results support the hypothesis that D1 receptor stimulation activates striatonigral neurons and modulates expression of AP-1-related genes in these neurons, while D2 receptor stimulation mediates tonic inhibition of AP-1 expression and activity in the striatopallidal neurons. Moreover, the findings provide evidence that the loss of dopaminergic input to the striatum, as occurs in Parkinson's disease, induces long-lasting alterations in the regulation of striatal gene expression which may contribute to the disease's progress.

Transcriptional regulation of c-Fes in myeloid leukemia cells.

The c-Fes proto-oncogene encodes a myeloid-specific protein-tyrosine kinase that is expressed preferentially in differentiated myeloid cells, but not in early myeloblast progenitor cells. To examine the basis for the phenotypic expression of c-Fes, the transcription initiation sites of the human c-Fes gene were mapped in myeloid leukemia cells and regulatory elements in the genomic c-Fes sequence were characterized. Two major transcription initiation sites were found in the myeloid leukemia cell line THP-1 which delineated exon 1 to be 72-83 bp. When the activity of the CAT reporter gene under the control of the c-Fes promoter region, untranslated exon 1 and intron 1 was measured in TF-1, K562 and MCF-7 cells, only TF-1 cells exhibited chloramphenicol acetyltransferase activity. In contrast, all cell lines supported reporter gene activity when intron 1 was deleted. Deletion analyses revealed a negative regulatory region in intron 1, which was localized by Southwestern analysis and DNA footprinting to a 14 bp region. This negative regulatory region suppressed reporter CAT activity in K562 and TF-1 cells when inserted downstream to the SV40 early promoter. These results suggest that the tissue-specific expression of c-Fes may result, in part, from the negative regulation of transcription in myeloid and nonmyeloid cells.

Transformations of Penicillium islandicum and Penicillium frequentans that produce anthraquinone-related compounds.

Wild-type strains of Penicillium islandicum and Penicillium frequentans, which produce anthraquinone and related compounds, were transformed to benomyl and hygromycin B resistance. Plasmids pSV50 and pBT6, with benomyl-resistant beta-tublin genes, and plasmids pAN7-1 and pDH25, with a bacterial hygromycin phosphotransferase gene under the control of Aspergillus nidulans sequences, were used respectively. Transformation frequencies with these plasmids were 10-20 transformants per micrograms of DNA per 4-8 x 10(7) viable protoplasts. Integration of plasmid DNAs into chromosomal DNAs was confirmed by Southern-blot analysis. Copy numbers and sites of integration varied among transformants. The integrated plasmid DNAs conferring a drug-resistant phenotype were mitotically stable with or without selection. The demonstration of such transformation systems in the essential first step in the application of recombinant DNA technology to study the biosynthetic genes of anthraquinone and related compounds in P. islandicum and P. frequentans.

[Effect of (-) stepholidine on dopamine turnover in various brain regions].

It has been shown before that DA antagonist (-) stepholidine [(-) SPD] changes the function of feedback regulation in the striatum. In order to compare the effect of the drug on other brain DA systems, the DA content and DOPAC level in brain areas or nuclei of rat were measured by HPLC-ECD. It was found that (-) SPD (10 mg/kg, ip) did not significantly affect the DA contents in the striatum, olfactory bulb, N. accumbens, substantia nigra, ventral tegmentum area, N. amygdala, hypothalamus, pituitary, piriform cortex, but did markedly increase the DOPAC levels in these brain areas or nuclei, and thus increase the DA turnover (DOPAC/DA). However, (-) SPD (10 mg/kg, ip) did not significantly affect either the DA contents or DOPAC levels in the prefrontal cortex and cingulate cortex, thus leaving the DA turnover in these areas unchanges. These results indicated that (-) SPD did affect the nigro-striatal, midbrain-limbic and hypothalamus-pituitary DA systems, but not the midbrain-cortex DA system. It is supposed that this difference may be due to the modulation by presynaptic DA autoreceptors in the major brain areas of DA nerve endings or neurosoma, but none in the cortex DA nerve endings.

Molecular cloning and heterologous expression of the gene encoding dihydrogeodin oxidase, a multicopper blue enzyme from Aspergillus terreus.

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzing the stereospecific phenol oxidative coupling reaction converting dihydrogeodin to (+)- geodin. We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-day-old mycelium of A. terreus, four clones were identified using anti-DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of the cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptide consists of 605 amino acids showing significant homology with multicopper blue proteins such as laccase and ascorbate oxidase. Four potential copper binding domains exist in DHGO polypeptide. The DHGO gene consists of seven exons separated by six short introns. Expression of the DHGO gene in Aspergillus nidulans under the starch or maltose-inducible Taka-amylase A promoter as an active enzyme established the functional identity of the gene. Also, introduction of the genomic DNA for DHGO into Penicillium frequentans led to the production of DHGO polypeptide as judged by Western blot analysis.

Dopamine receptor agonist potencies for inhibition of cell firing correlate with dopamine D3 receptor binding affinities.

The potencies for in vivo inhibition of substantia nigra pars compacta dopamine single cell firing were determined for apomorphine, BHT 920, N-0923, (+/-)-7-hydroxy-dipropylaminotetralin (7-OH-DPAT), (+)-3-(3-hydroxyphenyl)-N-propylpiperidine (3-PPP), pramipexole, quinelorane, quinpirole, RU 24926, U-86170, and U-91356. Significant correlation was obtained between the potencies of these 11 highly efficacious dopamine receptor agonists and the in vitro binding affinities at dopamine D3 receptors, but not at dopamine D2L receptors. These results support a functional role for the dopamine D3 receptor subtype in the autoreceptor-mediated regulation of dopamine cell activity, while a role for dopamine D2 receptors awaits further analysis. In addition, the results demonstrate the limitations of using currently available dopamine receptor agonists to delineate relative in vivo roles for the dopamine D2 and D3 receptor subtypes.

Electrophysiological effects of SKF 38393 in rats with reserpine treatment and 6-hydroxydopamine-induced nigrostriatal lesions reveal two types of plasticity in D1 dopamine receptor modulation of basal ganglia output.

To examine the role of D1 dopamine receptors in modulating basal ganglia output and how this role may be altered by changes in dopaminergic transmission, the effects of a D1 dopamine agonist on firing rates of substantia nigra pars reticulata (SNpr) neurons were determined in reserpine-treated rats and compared with effects observed in nigrostriatal-lesioned and normal rats. It was confirmed that systemic administration of the D1 dopamine agonist SKF 38393 (10 mg/kg i.v.) induces a small (averaging 20% above base line) increase in firing rates of SNpr neurons in normal rats and significantly inhibits SNpr single unit activity in rats studied 6 to 26 weeks after 6-hydroxydopamine (6-OHDA)-induced substantia nigra dopamine cell lesion. In contrast to results obtained after 6-OHDA lesions, SKF 38393 administration consistently and significantly increased the firing rates of SNpr neurons in rats treated for 6 days with reserpine (1 mg/kg/day s.c.); increases averaged 88% above base line. Five days after the 6-day reserpine treatment was discontinued, administration of SKF 38393 still induced a pronounced increase in SNpr activity. These increases were reversed by the administration of the D1 dopamine antagonist SCH 23390. SKF 38393 also significantly increased the firing rates of a subpopulation of SNpr neurons in rats treated with only a single dose of reserpine (10 mg/kg s.c. 4-7 hr). The difference between responses in reserpine and 6-OHDA-treated rats was not due to the shorter time course of the reserpine treatments, as SKF 38393 did not consistently affect the activity of SNpr neurons 7 days after 6-OHDA nigral dopamine cell lesion. When the subchronic reserpine treatment was administered to rats lesioned previously with 6-OHDA, D1 dopamine agonist-mediated inhibition of SNpr neuronal activity was obtained. The results show that SNpr responses to D1 dopamine agonist administration after reserpine treatment are enhanced ("supersensitive") relative to normal, whereas SNpr responses to D1 dopamine agonists in animals with 6-OHDA-induced dopamine cell degeneration are opposite to those observed in normal and in reserpinized animals. Current models of basal ganglia function predict that D1 dopamine agonists should reduce activity in the SNpr, because D1 dopamine receptors on striatonigral neurons are thought to stimulate striatonigral firing rates and to enhance the release of tau-aminobutyric acid in the SNpr. The present results call for modification of these concepts.